Graphene Quantum Dot Formulation for Cancer Imaging and Redox-Based Drug Delivery

Abstract

Treatment of complex conditions, such as cancer, has been substantially advanced by a field of molecular therapeutics. However, many of these therapies are limited by the dose toxicity and lack the predictive power of tomography-guided approaches. This can be in part addressed by targeting approaches, focusing the therapy on a tumor site. On the other hand, more benign gene therapeutics often require in vivo delivery, so as to avoid degradation in blood. Additionally, monitoring the location of the therapeutic can help evaluate its targeted accumulation and in part, efficacy. Therefore, there is an open need for multifunctional therapies allowing for targeted delivery, imaging, and treatment. Nanomaterial platforms can provide these capabilities, safely delivering therapeutics, concomitantly imaging their delivery pathways, and presenting sites for targeting agent attachment. Within this scope, the applications of nanoscale graphene derivatives have been largely studied. However, despite their remarkable properties, the biocompatibility and degradability of carbon-based platforms still raises a lot of debate, while modifying those with targeting agents may negatively affect their optical imaging properties. In order to address these issues, we develop graphene quantum dots (GQDs) produced via biocompatible bottom-up synthetic route as attractive candidates for bioimaging and targeted drug delivery. These GQDs are biocompatible and biodegradable, exhibit high-yield intrinsic fluorescence in the visible/near-infrared, high water solubility, pH-based fluorescence response, and have smaller size for more efficient cellular internalization. We explore these properties to develop GQD-based targeted delivery/imaging/treatment agents for cancer therapeutics. Our work utilizes nitrogen-doped GQDs as an emissive platform for covalent attachment of a targeting agent (hyaluronic acid (HA) targeted to the CD44 receptors on several cancer cell types) and oxidative stress-based cancer therapeutic (ferrocene (Fc)). The synthesized multifunctional formulation is characterized and its efficacy evaluated in vitro. Elemental mapping indicates that the purified from reactants synthetic product has an average iron content of 0.64 atomic percent, suggesting the successful attachment of the therapeutic, while FFT analysis of TEM images confirms the crystalline structure of the GQDs. Although GQDs alone yield no cytotoxicity as quantified via the MTT assay up to the maximum imaging concentrations of 1 mg/mL, the Fc-HA-GQD formulation exhibits a higher cytotoxic response in the cancer cells (MCF-7) targeted by the HA as opposed to healthy ones (HEK-293) that do not overexpress CD44 suggesting cancer-selective targeted efficacy. As Fc induces oxidative stress that is less mitigated in cancer cells, we expect it to also contribute to the observed cancer-selective treatment response. We further use spectrally-resolved in vitro fluorescence imaging showing the efficient cellular internalization maximized in MCF-7 cells over the HEK-293, and thus verifying the possibility of successful image-guided drug delivery. As a result, we propose Fc-HA-GQD formulation as a multifunctional targeted delivery, imaging, and cancer-specific treatment agent further to be studied in vivo.